Prolonged exposure of human fibroblasts to PGE results in a desensitization of adenylate cyclase. The enzyme exhibits a reduced response to not only PGE but to other effectors including cholera toxin. When membranes from control and desensitized cells are incubated with activated cholera toxin and [32P] NAD, however, the toxin catalyzes the ADP-ribosylation of the guanine nucleotide regulatory component of the cyclase equally well in both membranes. When adenylate cyclase is solubilized and analyzed by sucrose density gradient centrifugation, the activity from PGE - treated cells has an apparent lower molecular weight than the enzyme from control cells. PGE(1)-mediated desensitization may involve an uncoupling of the regulatory and catalytic components of adenylate cyclase. 2. In intact cells, cholera toxin activates adenylate cyclate after a lag period. Using an immunochemical procedure, bound toxin appears to be internalized during the lag period. In contrast, there is not degradation of the toxin during the lag and activiation periods. Generation of the A(1) subunit of the toxin parallels the activation of adenylate cyclase. Thus, intact cells first internalize cholera toxin and reduce it to form A(1), which can activate adenylate cyclase.